PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) is an acquired disorder of the hematopoietic stem cell. PNH is the result of a somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene. The product of this gene is crucial in the initial stage of synthesis of the glycosylphosphatidyl-inositol (GPI) anchor, a structure which attaches numerous important proteins to cell membranes. Because PIG-A gene mutation originates in a multipotent hematopoietic stem cell capable of differentiation, all progenitor cells deriving from the mutated HSC harbor the GPI defect, and have a complete or partial loss of expression of all GPI-anchored proteins (GPI-APs) on the cell surface. Some of these GPI-anchored proteins, such as CD55 and CD59, play important roles in the control of complement. Activated serum complement is known to play a crucial role in the hemolytic anemia. In normal individuals, red blood cells are protected from complement-mediated destruction, whereas those deficient in anchored proteins CD55 and CD59 undergo hemolysis, predominantly intravascular. Expansion of PNH type clones is commonly associated with idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS), conditions of immune-mediated failure of normal hematopoiesis. In was shown that in AA, up to 60% of patients harbor PNH-type cells. In MDS, approximately 15% of patients harbor PNH-type cells. Diagnosis and follow-up of PNH patients is currently done by flow cytometry (FC)-based assays involving evaluation of deficient expression of GPI-APs in PB cells. Besides testing the expression of the two GPI-APs CD55 and CD59 on both red cells and neutrophils, a newer and more sensitive approach for PNH testing emerged, involving fluorescently labeled inactive variant of the protein aerolysin (FLAER). PB red and white cells have been extensively studied in PNH. Less has been done to assess the abnormalities of BM populations in these patients. BM specimens are generally considered less suitable than PB owing to variable expression of GPI-APs during the various stages of hematopoietic cell maturation and for that reason, are rarely studied for PNH evaluation. However, BM aspirates from patients with unexplained cytopenias, including bone marrow failure syndromes, are frequently submitted to laboratories for general diagnostic purposes. Specific goals of this project: 1. Analysis of fluorescently labeled aerolysin (FLAER) binding to bone marrow (BM) cell populations in normal volunteers and patients with BM failure conditions such as aplastic anemia (AA) or myelodysplastic syndrome (MDS) who have detectable PNH in the peripheral blood (PB). 2. Comparison of PNH clone size in BM and PB within the same cell types. 3. Detection of PNH in BM by combinations of antibodies routinely used for diagnosis of BM disorders in clinical laboratories. 4. Assessment of proliferation and apoptosis of PNH cells and normal cells in the same BM samples. Methods: Flow cytometry analysis of FLAER binding, and expression of CD55/CD59 in conjunction with combinations of other antibodies in BM cells. Proliferation and apoptosis assays by flow cytometry. Results: FLAER binds to all normal BM cells, except for erythrocytes, which bind CD55 and CD59. Within each lineage except for RBCs, FLAER binding increases with cell maturation and reaches the highest level on mature elements. In PNH, the defect (clone size) is smaller in lymphocytes than in other BM cells. PNH clone sizes in BM and PB are very similar, making BM equally suitable for PNH detection and quantitation. With combination of antibodies routinely used for general diagnosis (anti CD45, CD13, CD11b, CD64 and the anchored proteins CD16 and CD14) PNH clones can also be detected in BM mature neutrophils and monocytes with high specificity and sensitivity. Currently, data continue to be analyzed to answer the question of predicting variation in clone size (which has therapeutic implications)based on longitudinal analysis of clone sizes at prior values. PRESENTATIONS 1. Dulau Florea A, Young NS, Maric I, Braylan RC. Human Pathology (Poster Abstract). Expression of Glycosylphosphatidylinositol (GPI) Anchor Protein (AP) in Bone Marrows of Normal Subjects and Aplastic Anemia Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).Human Pathology (Poster abstract).The USCAP Annual Meeting 2016, Seattle, WA (March 12-18, 2016). 2. Dulau Florea A, Young NS, Maric I, Jordan EK, Jiang C, Ahmad F, Braylan R. Immunophenotypic Abnormalities Highly Suggestive of Paroxysmal Nocturnal Hemoglobinuria (PNH) can be Detected with Routine Flow Cytometric Analysis of Bone Marrow (BM). Human Pathology (Poster abstract).The USCAP Annual Meeting 2017, San Antonio, TX (March 4-10, 2017). 3. Dulau Florea A, Young NS, Jordan E, Maric I, Braylan R. 154 Bone Marrow Aspirate Samples are Equal to Peripheral Blood for the Detection of Paroxysmal Nocturnal Hemoglobinuria (PNH). Leuk Res. April 2017, Vol.55: S96 (Poster abstract). The 14th International Symposium on Myelodysplastic Syndromes (MDS 2017), Valencia, Spain (May 3-6, 2017).